WO2012022032A1

WO2012022032A1 - Endogenous antiviral complex-probiotic-preparation for prawn

Info

Publication number: WO2012022032A1
Application number: PCT/CN2010/076082
Authority: WO
Inventors: 宋晓玲; 黄倢; 杨冰; 刘莉
Original assignee: 中国水产科学研究院黄海水产研究所
Priority date: 2010-08-17
Filing date: 2010-08-17
Publication date: 2012-02-23
Also published as: CN102740707B; CN102740707A

Abstract

An endogenous antiviral complex-probiotic-preparation for prawn. The preparation consists of 5-20% by weight of Bacillus firmus SSL065 powder, 5-10% by weight of Vibrio alginolyticus SSL073 powder and 70-90% by weight of carriers. The two bacteria components have synergistic effect. The present preparation has multiple biological functions such as obvious improvement of cultured prawn's nonspecific immunity level, great reinforcement of cultured prawn's resistance to pathogenic bacteria infection, and especially significant improvement cultured prawn's resistance to viral pathogen infection.

Description

Endogenous shrimp antiviral compound probiotic preparation

Technical field

The invention belongs to the field of probiotic preparations, and particularly relates to an endogenous anti-virus compound probiotic preparation of shrimp.

Background technique

There are a large number of bacterial microbes in the shrimp culture environment. They are not only an important part of the shrimp food chain/net, but also participate in the material circulation and energy flow in the culture system, and play an important role in maintaining ecological balance and optimizing environmental quality. An important role. Bacteria in the aquatic environment enter the digestive tract through the feeding behavior of the shrimp. Different kinds of bacteria form a complex and dynamic microbial ecosystem in the digestive tract of the shrimp, whose structure and composition are controlled by the biological body and external environmental factors. The main functions of the beneficial bacteria in the digestive tract include: maintaining the micro-ecological balance of the digestive tract, improving the digestion ability of the feed, enhancing the non-specific immunity of the body, and preventing the proliferation and spread of pathogenic microorganisms in the digestive tract. However, the development and utilization of disease-resistant microorganisms in aquaculture environments, especially those with antiviral effects, have rarely been reported.

The occurrence and prevalence of viral diseases is one of the major problems plaguing shrimp farming. The main cause of the virus-borne pathogens is the white spot syndrome virus (WSSV). The prawn outbreak caused by the virus began to spread in Asia in 1993 and then spread to the American shrimp culture area. It is still the most serious disease in shrimp farming worldwide. The disease is characterized by rapid onset, rapid death, and high mortality. Every year, the global shrimp production is reduced by half due to the occurrence and spread of WSSV. The annual loss caused by the disease in China is as high as 3 to 5 billion yuan. Taura syndrome virus (TSV) also poses a major threat to shrimp during culture. Infectious hypodermal & haematopoietic necrosis virus (IHHNV) Hepato pancereatic parvovirus (HPV) ) also caused different degrees of damage in the shrimp breeding period and juvenile shrimp period. Because the virus has strict cell parasitic properties, there is no effective chemical drug to prevent and treat shrimp virus disease; and the shrimp lacks a specific immune system, and the vaccine cannot effectively control the effect. Therefore, the search for effective ecological control measures, especially endogenous bacterial preparations that enhance the disease resistance of shrimp, has become the most promising strategy for controlling shrimp virus diseases.

Summary of the invention

The object of the present invention is to provide a method for enhancing the immune level of shrimp cells and body fluids, especially for improving the anti-viral infection ability of shrimps, and the like, and the invention can significantly improve the anti-viral infection ability of shrimps. An endogenous prawns antiviral compound probiotic preparation for the digestive tract of shrimp.

The information about the two strains involved in the present invention is as follows:

The strain Bacillus firmus SSL065 (Bacillus firmus) was isolated from the digestive tract of healthy Chinese shrimp, and was determined by high-dose injection or feeding experiments for Chinese traits (3⁄4«<3⁄43⁄4s chinensis), under J onCTMeMS vannamei, Japanese prawns are safe and pathogenic. The strain was cultivated in Wuhan China on July 30, 2010. Deposited by the Tibet Center, the deposit number is: CCTCC M 2010192. The 16 rDNA base sequence of the Bacillus lentus SSL065 strain is shown in SEQ ID NO: 1.

Vibrio alginolyticus strain SSL073 (Vibrio alginolyticus) strain was isolated from the digestive tract of healthy Chinese shrimp. It was determined to be safe and pathogenic to Chinese shrimp, P. vannamei, and Japanese prawns by high-dose injection or feeding experiments. The strain was deposited at the China Center for Type Culture Collection in Wuhan on July 30, 2010. The deposit number is: CCTCC M 2010193. The 16 rDNA base sequence of the Vibrio alginolyticus SSL073 strain is shown in SEQ ID NO: 2.

The endogenous anti-virus compound probiotic preparation of the invention is characterized in that: the total mass percentage is composed of Bacillus lentus SSL065 powder, Vibrio alginolyticus SSL073 powder and auxiliary protective carrier: Bacillus sp. SSL065 powder 5-20%, Vibrio alginolyticus SSL073 powder 5-10%, and auxiliary protective carrier 70-90%.

The Bacillus lentus SSL065 powder is prepared by freeze-drying, wherein the concentration of Bacillus lentus SSL065 is up to ^ ^) ¹ ^?!; / gram; the Vibrio alginolyticus SSL073 powder is spray-dried, no Vibrio alginolyticus SSL073 can be detected.

The auxiliary protective carrier may be one or more selected from the group consisting of dextran powder, wheat dextrin, and corn starch.

The preparation method of Bacillus lentus SSL065 powder is as follows: a small amount of scribble is collected from the strain stored at -80 °C in 2216E solid medium, and cultured at 37 ° C for 24 hours to obtain a single colony, and a single colony is inoculated to the seed of Bacillus lentus. In the culture medium, 37 °. The seed solution was obtained by shaking the flask for 24 hours, and then the seed solution was inoculated into the fermentation broth of Bacillus lentus, and the fermentation temperature was controlled at 30-38 ° C. When the OD _{6 (K)} value of the fermentation broth reached 1.5 2.0, the fermentation was terminated. The fermentation broth is obtained. The fermented bacteria liquid was centrifuged and concentrated 80-100 times to obtain the bacterial sludge, and a commercially available trehalose protecting agent having a mass ratio of 6% was added to the slime, uniformly mixed, and then freeze-dried to obtain Bacillus lentus SSL065 powder.

Among them, the percentage of the components of Bacillus lentus seed culture solution is: dextrin 1.5%, peptone 0.5%, ferrous sulfate 0.05%. ; pH is 5.7.

The percentage by mass of the fermentation broth of Bacillus lentus is: peptone 0.5%, yeast extract 0.1%, ferrous sulfate 0.02% ₀ .

The preparation method of Vibrio alginolyticus SSL073 powder is as follows: a small amount of scribble is collected from the strain stored at -80 °C in 2216E solid medium, and cultured at 28 °C for 24 hours to obtain a single colony, and a single colony is inoculated into Vibrio alginolyticus. In the seed culture solution, the seed liquid was obtained by shaking in a shake flask at 28 ° C for 24 hours, and then the seed liquid was inoculated into the fermentation broth of Vibrio alginolyticus to ferment and culture, the fermentation temperature was controlled at 25-28 ° C; when the OD600 value of the fermentation liquid reached 1.6, Stop the fermentation; obtain the fermentation broth; take the fermentation broth and centrifuge to concentrate 20-50 times to prepare the turbid liquid. According to the volume ratio of bacteria turbid liquid 1: 5-1: 10, the cells were resuspended in the lysate, digested at 37 ° C for 24 hours; 300 W, I OS / I OS ultrasonically disrupted for 20 minutes, the sprayed bacteria were spray dried (into the air) The Vibrio alginolyticus SSL073 powder was prepared at a temperature of 195 ° C and 85 ° C, respectively.

The mass percentage of the above-mentioned Vibrio alginolyticus seed culture solution is: glucose 1.2%, peptone 1.0%, beef extract 0.3%, yeast extract 0.5%, and trisodium citrate 0.8%. , K ₂ HPO ₄ 0.2°/oo KH ₂ PO ₄ 0.1%. , MgSO ₄ 0.4%. ; pH is 7.2.

2 The above-mentioned mass percentage of the fermentation broth of the Vibrio alginolyticus is 1.2% of glucose, 0.2% of peptone, 1.5% of beef extract, and 2.5% of NaCl.

The above lysate was: 50 mM Tris-HCl, pH 8.0; 2 mM EDTA, 100 mM NaCl, 0.1% Triton X-100, plus lysozyme to 10 (^g/ml).

The composite probiotic preparation of the invention is a composite of Bacillus licheniformis SSL065 live bacteria and Vibrio alginolyticus SSL073 component, and the living bacteria and bacterial components contained in the invention are mutually promoted and synergistically, and the non-specificity of the cultured shrimp is obviously improved. The level of immunity greatly enhances the resistance of cultured shrimps to pathogens, and in particular, can significantly improve the biological functions of shrimps against viral pathogens.

detailed description

Example 1:

1. Preparation of the composite probiotic preparation of the present invention:

The preparation method of Bacillus lentus SSL065 powder is as follows: pick a small amount of the solid medium from the strain stored at -80 °C, and incubate it at 37 °C for 24 hours to obtain a single bacteria, inoculate it in the seed solution, shake flask culture, and then The seed solution is inoculated into the fermentation broth and fermented, and the fermentation temperature is controlled at 30-38 ° C. When the 0D _{6 (K)} value of the fermentation broth reaches 1.6, the fermentation is terminated, and the fermentation broth is obtained. The fermentation broth is centrifuged and concentrated 80-100 times to obtain the bacterial sludge, and the mass ratio of 6% trehalose protecting agent is added to the slime, uniformly mixed, and then freeze-dried to obtain a living bacteria concentration higher than 10 ¹² CFU/g. Bacillus lentus SSL065 powder.

The preparation method of Vibrio alginolyticus SSL073 powder is as follows: a small amount of scribble is collected from the strain stored at -80 ° C in 2216E solid medium, and cultured at 28 ° C for 24 hours to obtain a single colony, and then inoculated into the seed liquid in a shake flask culture. The seed solution is inoculated into the fermentation broth and fermented, and the fermentation temperature is controlled at 25-28 ° C. When the 0D _6QQ value of the fermentation broth reaches 1.6, the fermentation is terminated; the fermentation broth is obtained; and the fermentation broth is centrifuged to concentrate 20-50 times. The turbid liquid was prepared. The bacteria were resuspended in the lysate according to the volume of the turbid liquid, 1/5-1/10, and digested at 37 °C for 24 hours; 300w, 10s/10s ultrasonically disrupted for 20 minutes, and the broken bacteria solution was spray dried (into the wind and out The air temperature was 195 ° C and 85 ° C, respectively, to prepare Vibrio alginolyticus SSL073 powder.

20 g of Bacillus subtilis powder, 5 g of Vibrio alginolytic powder, and 75 g of auxiliary protective carrier corn starch were uniformly mixed to prepare a composite probiotic preparation.

2, the effect detection of compound probiotics:

The compound probiotic preparation is added to 10 kg of basic shrimp feed (including fishmeal 30%, shrimp powder 10%, soybean powder 25%, peanut powder 25%, flour 3%, corn flour 3%, multivitamin 1%). Formulated as an immunofeed containing a probiotic preparation. The various raw materials were crushed through a 60-mesh sieve, accurately weighed and thoroughly mixed step by step, using sodium alginate as a binder, and an appropriate amount of water was extruded into a pellet feed with a small meat grinder, at 40 ° C. Dry to a moisture content of less than 10%, package in a bag, and store in a refrigerator at 4 °C.

There were more than 600 tails of P. vannamei, and the initial body length of the experimental prawns was (5.70± 1.18) cm. It was randomly divided into two treatment groups: the experimental group and the control group. Each treatment group had 3 parallels, and each parallel group stocked the shrimp 60. tail. Change water once a day, change water every day

3 The amount is 1/3; the feed is fed 4 times a day, the daily feed amount is about 0.9-1.2 g/10 tails, the remaining feed is sucked after feeding for lh, and the amount of feed is adjusted according to the amount of residual bait; The experimental group used the interval feeding method, that is, feeding the immune feed for 4 consecutive days, feeding the basic feed for 3 days, and circulating for 7 days until the end of the experiment. The water temperature is 24-26 ° C, continuous inflation, and the nurse is kept for 7 days before the experiment. The control group was fed the basal feed throughout the experimental period. Sampling was performed at 0, 5, 10, 15 and 20d, respectively, to determine the effect of the anti-disease microbial preparation on the non-specific immunity level of L. vannamei. The results showed that: compared with the control group, the anti-disease microbial preparation can significantly increase the activity of acid-related enzymes such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme in the serum of shrimp, ie, the shrimp is improved. The level of immunity (Table 1).

Table 1 Addition of anti-disease microbial preparations to the serum immune enzyme activity of Penaeus vannamei

Example 2:

1. Preparation of compound probiotics:

The preparation method of Bacillus lentus SSL065 powder and Vibrio alginolyticus SSL073 powder is the same as that in the first embodiment, 5 g of Bacillus lentus SSL065 powder, 10 g of Vibrio alginolyticus SSL073 powder, 65 g of auxiliary protective carrier corn starch, dextran 20 g of powder was uniformly mixed to prepare a composite probiotic preparation.

2, the effect detection of compound probiotics:

Add the compound probiotic preparation to 10 kg of basic shrimp feed ingredients (including fishmeal 30%, shrimp powder 10%, soy flour 25%, peanut powder 25%, flour 3%, corn flour 3%, multivitamin 1%) , formulated as an immune feed containing anti-disease microbial preparations. The various raw materials were crushed through a 60-mesh sieve, accurately weighed and thoroughly mixed step by step, using sodium alginate as a binder.

4 Add appropriate amount of water and squeeze it into pelleted feed with a small meat grinder. Dry it to a moisture content of 10% or less at 40 °C, pack it in a bag, and store it in a refrigerator at 4 °C.

More than 800 tails of P. vannamei, body length (6.80 ± 0.38) cm, were randomly placed in a 550 L FRP sink. Change the water once a day, the daily water exchange rate is 1/3; feed the feed 4 times a day, the daily feed amount is about 1.5-2.0 g/10 tail, after feeding for lh, the remaining feed is sucked, and according to the residual bait How much to adjust the amount of feed; water temperature 22 ~ 25 ° C, pH 8.2 ± 0.2, and continuous inflation, held for 7 days before the experiment.

After the holding period, the experimental shrimps were randomly divided into two groups: the experimental group and the control group. Each treatment group had 3 parallels, and each parallel group stocked 90 shrimps. The control group was fed the basal feed throughout the experimental period. The experimental group was fed with the immune feed at intervals, that is, the basal feed was fed for 1 day, and the immune feed was administered for 1 day until the end of the experiment. On the 22nd day after feeding the immunized feed, the shrimp disease infected with the white spot syndrome virus was directly fed at a dose of 4.2 g/kg body weight, and the number of prawns that died daily during the virus infection stage was recorded, and the WSSV infection was calculated within 14 days. Cumulative mortality of Penaeus vannamei. The results showed that compared with the control group, the anti-disease microbial preparation significantly reduced the mortality of WSSV infection in shrimp, and the immune protection rate reached 65.52%, which improved the anti-WSSV infection ability of shrimp (Table 2).

Table 2 Cumulative mortality of Penaeus vannamei after WSSV challenge (%)

Time of infection (days) Mean cumulative mortality in the immunized group (%) Average cumulative mortality in the control group (%)

1 0. 00 0. 00

2 0. 00 8. 33

3 0. 00 8. 33

4 0. 00 11. 67

5 3. 33 15. 00

6 3. 33 15. 00

7 3. 33 38. 33

8 8. 33 63. 33

9 18. 33 81. 67

10 23. 33 91. 67

11 28. 33 95. 00

12 33. 33 96. 67

13 33. 33 96. 67

14 33. 33 96. 67

Example 3:

Application effect of antiviral compound probiotics in the production of shrimp shrimp culture:

The shrimp anti-virus compound probiotic preparation (containing 5-20% of Bacillus subtilis powder, 5-10% of Vibrio alginolytic powder, 70-90% of auxiliary protective carrier) is uniformly mixed and added to ordinary shrimp feed. Granulated feed (41% crude protein, 4-8% crude fat, 5% crude fiber, 16% coarse ash, Ca ²⁺ l-3%, total phosphorus 1-2%, moisture 11%). The test was arranged in Shanghai Jinshan Breeding Company, and 6 rectangular earthen ponds were selected, each about 7 mu. The cultured species was Penaeus vannamei and the trial began on July 5. Three test cells were fed, and each time the immune feed was fed at 6 am and 6 pm, After 7 days of feeding of the immunized feed, the feed of the control shrimp was fed for 7 days, and this was repeated until the end of the culture test on September 16; the control pool was 3 test cells, and the feeding time was 6 am and 6 pm every day, and Feed the common prawn feed throughout. From the beginning of the culture until the end of August, all the culture ponds including the test ponds and the control ponds had good growth of the shrimps, and the adjacent cultured shrimps had a considerable part of the disease in early August. By the end of August, due to the intensification of the adjacent shrimp ponds, the seabirds and other seabirds preyed to spread horizontally, which caused the experiment to begin to be affected. In particular, the control pool began to develop disease. The detection of the nucleic acid probe confirmed that the pathogen was the white spot syndrome virus. . One of the pools was the most damaged and the survival rate was less than 20%. The incidence of the test pool was significantly lighter, and only individual dead shrimps were found. By September 16, as the incidence of the control pool continued, it was decided to end the trial and collect shrimp. From the test results (Table 3), we can see:

Table 3 Comparison of the application of antiviral compound probiotics in the production of shrimp shrimp

The average survival rate of shrimp in the experimental group was 65.4%, which was 34.6% higher than that of the other feeding ponds. The survival rate was significantly different (P<0.01). By the end of the breeding period, the average yield per hectare in the experimental pool was 8084.9Kg, the output increased by 33.4%, and the economic benefits were remarkable. The results showed that: shrimp anti-disease microbial preparation has a good preventive effect on viral infection.

Industrial applicability

The composite probiotic preparation of the invention is a composite of Bacillus licheniformis SSL065 live bacteria and Vibrio alginolyticus SSL073 component, and the living bacteria and bacterial components contained in the invention are mutually promoted and synergistically, and the non-specificity of the cultured shrimp is obviously improved. The level of immunity greatly enhances the resistance of cultured shrimp to pathogens. The composite probiotic preparation can be used in the production of shrimp shrimp culture to enhance the immune level of shrimp cells and body fluids.

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Claims

QuanheiJ request letter

1. An endogenous shrimp antiviral compound probiotic preparation, characterized in that the compound probiotic preparation is mixed with Bacillus firmus SSL065 powder, Vibrio alginolyticus SSL073 powder, and auxiliary protective carrier; its total mass percentage It is: Bacillus firmus SSL065 powder 5-20%, Vibrio alginolyticus SSL073 powder 5-10%, auxiliary protection carrier 70-90%.

2. An endogenous shrimp antiviral compound probiotic preparation as claimed in claim 1, characterized in that the concentration of viable Bacillus firmus SSL065 bacteria in the above-mentioned Bacillus firmus SSL065 bacterial powder is 10 ¹² CFU/g.

3. An endogenous shrimp antiviral compound probiotic preparation as claimed in claim 1, characterized in that the above-mentioned auxiliary protection carrier is one or more of dextran powder, wheat dextrin and corn starch. .

4. An endogenous shrimp antiviral compound probiotic preparation as claimed in claim 1, characterized in that the preparation steps of the above-mentioned Bacillus firmus SSL065 bacteria powder are as follows: Divide the bacteria stored at -80°C. Line on 2216E solid medium, culture at 37°C for 24 hours to obtain a single colony, inoculate a single colony into the Bacillus firmus seed culture solution, culture it in a shake flask at 37°C to obtain the seed solution, and then inoculate the seed solution into Bacillus firmus Fermentation culture in the fermentation broth, the fermentation temperature is controlled at 30-38°C _; when the 0D _6QQ value of the fermentation broth reaches

At 1.5 to 2.0 hours, the fermentation is terminated to obtain the fermented bacterial liquid; the fermented bacterial liquid is centrifugally concentrated 80-100 times to obtain bacterial mud, and a trehalose protective agent with a mass ratio of 6% is added to the bacterial mud and mixed evenly. Then freeze-drying is carried out to prepare Bacillus firmus SSL065 bacterial powder.

5. An endogenous shrimp antiviral compound probiotic preparation as claimed in claim 4, characterized in that the mass percentage of the above-mentioned Bacillus firmus seed culture liquid ingredients is: dextrin 1.5%, peptone 0.5%, sulfate Iron 0.05%. , pH value is 5.7.

6. An endogenous shrimp antiviral compound probiotic preparation as claimed in claim 4, characterized in that the mass percentage of the above-mentioned Bacillus firmus fermentation liquid ingredients is: peptone 0.5%, yeast extract 0.1%, sulfate Iron 0.02%. .

7. An endogenous prawn antiviral compound probiotic preparation as claimed in claim 1, characterized in that the preparation steps of the above-mentioned Vibrio alginolyticus SSL073 powder are as follows: From strains stored at -80°C Pick a small amount and streak it on 2216E solid medium, culture it at 28°C for 24 hours to obtain a single colony, inoculate a single colony into the Vibrio alginolyticus seed culture solution, culture it in a shake flask at 28°C for 24 hours to obtain the seed solution, and then inoculate the seeds. The liquid is inoculated into the Vibrio alginolyticus fermentation broth for fermentation culture, and the fermentation temperature is controlled at 25-28°C _; when the OD _6QQ value of the fermentation broth reaches 1.6, the fermentation is terminated to obtain the fermentation bacterial liquid; the fermentation bacterial liquid is centrifuged and concentrated for 20- 50 times to prepare bacterial turbid liquid; Add lysis solution to resuspend the bacteria according to the volume ratio of bacterial turbid liquid 1:5-1:10, digest at 37°C for 24 hours; ultrasonically crush at 300w, 10s/10s for 20 minutes, and spray-dry the crushed bacterial liquid to prepare algae. Bacteria SSL073 powder.

8. An endogenous prawn antiviral compound probiotic preparation as claimed in claim 7, characterized in that the above-mentioned Vibrio alginolyticus seed culture liquid ingredient mass percentage is: glucose 1.2%, peptone 1.0%, beef extract 0.3%, yeast extract 0.5%, trisodium citrate 0.8%. , K ₂ HPO ₄ 0.2%. , K¾PO ₄ 0.1% _o , MgSO ₄ 0.4% o, pH is 7.2.

9. An endogenous shrimp antiviral compound probiotic preparation according to claim 7, characterized in that the mass percentage of the above-mentioned Vibrio alginolyticus fermentation liquid ingredients is: glucose 1.2%, peptone 0.2%, beef extract 1.5 % and NaC1 2.5%.

10. An endogenous shrimp antiviral compound probiotic preparation as claimed in claim 7, characterized in that the above-mentioned lysate is: 50mM Tris-HCl, pH8.0; 2mM EDTA, 100mM NaCl, and 0.1% Triton X-100, add lysozyme to 100 g/mL

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